Helicobacter pylori is a newly discovered bacterium found in the upper gastrointestinal tract of humans. Warren, J. R., Marshall, B. J. "Unidentified curved bacilli on gastric epithelium in active chronic gastritis." Lancet, 1:1273-1275 (1983). The bacterium was originally named Campylobacter pylori because it appeared to be a microaerophilic, spiral, gram-negative bacterium with the guanine and cytosine content normally found in the Campylobacter genus. However, it was later transferred to the Helicobacter genus when its ultrastructure and fatty acid composition were found to be very different from that of Campylobacters. "Campylobacter pylori becomes Helicobacter pylori." (editorial). Lancet, 2:1019-20 (1989). Its presence is associated with acute and chronic inflammation in the gastric antrum, gastritis and peptic ulcer disease. Blaser, M. J. "Gastric Campylobacter-like organisms." Gastroenterology, 93:371-383 (1987). In addition, the presence of H. pylori is an important marker for various inflammatory conditions of the upper gastrointestinal tract. Rathbone, B. J. et al. "Campylobacter pyloridis--a new factor in peptic ulcer disease?" Gut, 27:635-641 (1986). Detection of H. pylori can be achieved by many different techniques using either invasive or noninvasive methods. However, all of these current methods are flawed and/or inconvenient for the various reasons discussed below.
The current invasive methods require gastric biopsies and include culture, histology, and detection of preformed bacterial enzymes. Unfortunately, however, these methods are both time-consuming and uncomfortable for the patient. In addition, false-negative cultures are common--especially early on in the learning curve of laboratories. Hazell, S. L., Graham, D. Y. "Campylobacter pylori in Perspective." Practical Gastroenterology, Vol. XII, No. 7, pp. 11-15 (1988). In some instances, detection of the bacterium may be difficult because of a patchy distribution of H. pylori growth in the gastric mucosa. Moreover, failure to culture the organism may result from swallowed anesthetic, simethicone (as used during endoscopy), previous use of antibiotics, use of H.sub.2 -receptor antagonists, contamination of biopsy forceps with glutaraldehyde, biopsy of nongastric mucosa, and poor handling of or delay in plating specimens. Dooley, C. P., Cohen, H. "The Clinical Significance of Campylobacter pylori." Annals of Internal Medicine, 108:70-79 (1988). Many practitioners also work in clinics where culture procedures are not practicable, making alternative methods of detection more useful. Id. Hence, non-invasive methods were developed as an attempt to circumvent these problems, and to provide more useful alternative methods of detection.
Noninvasive procedures used to detect the presence of H. pylori fall into two groups: gastric urease detection by means of a breath test, and serology, where specific antibody detection systems are used to identify those individuals infected by the bacterium. Like the invasive procedures, however, the noninvasive procedures are also encumbered with problems.
Helicobacter pylori produces urease, an enzyme that hydrolyzes urea to form CO.sub.2 and ammonia with a concomitant alkaline shift in pH. In the urea breath test, the patient is given .sup.13 C- or .sup.14 C- labelled urea with a beverage. The interaction of urease with the administered urea produces CO.sub.2 --which being membrane soluble, passes across the mucosa into the blood and into the breath where its concentration can be determined. Although this test can reliably detect an active H. pylori infection, it has many disadvantages including, inconvenience, cost in both materials and time, skilled personnel are required to perform the test, and the undesirable radiation exposure associated with the use of .sup.14 C-urea.
As a result, investigations with human serum were undertaken to replace the inconvenient and expensive procedures described above. The investigations revealed that H. pylori infections mediated a significant circulating antibody response against this organism; however, these antibody responses can be extremely inconsistent. Jones, D. "Campylobacter pyloridis serology." Serodiagn. Immunother. Infec. Dis., 1:87:89 (1987).
Since the discovery of H. pylori. extensive studies have been performed on its antigens, and a wide variety of these antigens are now used for detection of H. pylori antibodies in serum. Immunoblotting studies have also demonstrated several major protein antigens that are detected by most sera. These include two urease-associated proteins (63 and 56 Kilodaltons (kDa)), a putative flagellin (54 kDa), and 116 and 48 kDa surface proteins and a 31 kDa outer membrane protein. von Wuffen, H. et al. "Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations." J. Clin. Pathol., 41: 653-659 (1988). In fact, U.S. Pat. No. 4,882,271 to Evans et al. describes a serum assay for the detection of Campylobacter pylori using the urease-associated antigens. Unfortunately, a number of these proteins share antigenic determinants with other Campylobacter species, most notably the putative flagellin, which results in a lack of specificity in the current serum assays. A particularly interesting protein of 116 kDa, however, appears to be H. pylori specific. Id. Table 1 summarizes the molecular weights of antigen fractions of H. pylori which have been reported.
TABLE 1 ______________________________________ Helicobacter pylori Antigens Antiqens (kDa) ______________________________________ 128 120 100-120 100-110 92 84 82 58-66 60 33 31 29 24 22 20 14-21 ______________________________________
Current serological techniques to determine antibody responses against H. pylori include hemagglutination, bacterial agglutination, complement fixation and enzyme-linked immunosorbent assays (ELISA). ELISA appears to be the most sensitive, easiest and convenient of the test systems and is, therefore, the most commonly used. However, major problems have been encountered with the ELISA involving the source, type and characteristics of the antigen used. Newell, D. G., Rathbone, B. J. "Review Article: The serodiagnosis of Campylobacter pylori infection." Serodiaon. Immunother. Infec. Dis., 3:1-6 (1989).
The literature reveals a tug-of-war between sensitivity and specificity, with adequate sensitivity producing inadequate specificity and vice versa. Studies using multiple antigens indicated that some sera from H. pylori-colonized patients could be negative against antigen from one strain but positive when tested against an antigen derived from several strains. Id. In addition, there is also an interspecies antigenic cross-reactivity. The putative flagellar protein of H. pylori, (molecular weight of about 60 kDa), which appears to be amongst the immunodominant antigens, has antigenic identity with the flagella of other Campylobacter species, especially the thermophilic group C. jejuni and C. coli. Id. Cross-reactivity appears to occur with the 31 kDa antigen as well. In addition to cross-reactivity, studies have demonstrated a strain variation among the H. pylori outer membrane antigens. As a result, until the present invention, a mixture of H. pylori antigens suitable for inclusion in an antigenic preparation for ELISA, that maintains both sensitivity and specificity, has been elusive.
EPO application No. 0329510 to Martin J. Blaser, purports to have discovered an antigenic composition which overcomes this tug-of-war. The antigenic composition described in that application comprises a mixture of fragments from five (5) strains of H. pylori which is enriched with at least one of the following antigens: 63, 57, 45 and 31 kDa fragments. However, the enriched presence of the cross-reactive 63 and 31 kDa fragments significantly impairs the specificity of this assay. Consequently, there is still a need for an antigenic composition which is at the same time, both highly specific and highly sensitive for antibodies directed against H. pylori.
In addition to the antigen-associated problems, the handling of blood and blood products poses significant biological hazards. Tests based on the detection of antibodies in serum involves blood collection and blood component separation procedures. While these procedures may not be complicated, they involve a degree of biological hazard, which includes needle sticks and contamination with blood-borne infectious agents. Because of this hazard, laboratory personnel prefer assays involving urine samples which provide greater hygienic protection against these risks.
With this in mind, it has been established that normal urine contains significant amounts of both IgG and IgA. There also appears to be a good correlation between levels of H. pylori-specific IgG in serum and in urine in infected individuals even though the immunoglobulin titer in urine is lower than in serum. Because the immunoglobulin titer is lower in urine than in serum, assays with low sensitivity may not be able to detect antibodies present in dilute urine samples. Hence, neither the Evans nor Blaser assays discussed above could produce reliable results in a urine sample.
In addition, urine can easily be obtained and procedures involving urine collection and testing are simple and essentially void of biological risk. Moreover, a urine assay provides laboratory personnel greater hygienic protection against the biological hazards associated with handling blood and blood-related products. Hence, there is a definite need for a highly specific and highly sensitive diagnostic test for detecting antibodies directed to H. pylori in a specimen which overcomes the problems discussed above. The present invention meets this need and provides related advantages as well.